As is well known in the art, genetic information is encoded in the structure of DNA molecules (genes). Expression of the encoded information involves a two-part process. In a process known as transcription, mRNA is synthesized from the DNA template. The mRNA carries the genetic code transcribed from that DNA to specialized complexes within a living cell known as ribosomes. In a subsequent process known as translation, the cell's ribosomes "read" the mRNA "message" to produce a protein composed of a sequence of amino acids corresponding to the sequence of base pairs present in the mRNA. The resulting protein is the gene product coded by that DNA and mRNA.
It is also known that eukaryotic genes commonly contain coding regions, called exons, interrupted by non-coding regions, called intervening sequences or introns. It has been found that the RNA molecules transcribed from such "split" genes containing introns are longer than the mRNAs that subsequently produce the protein specified by the gene. Thus, by a process called splicing, the introns are excised from the newly synthesized, split precursor transcript and the exons are ligated together to form an unbroken fully-coding mRNA (Breathnach and Chambon, Ann. Rev. Biochem. 50, 349 [1981]).
There are also a few reports of introns within prokaryotic DNA, but split genes are relatively rare in bacterial species. The thymidylate synthase (td) gene of bacteriophage T4 was the first prokaryotic protein-coding gene shown to have an intervening sequence (Chu et al., Proc. Natl. Acad. Sci. USA 81, 3049 [1984]). The configuration of the td gene of bacteriophage T4 containing a 1 kilobase (kb) intron is disclosed by Chu et al., Proc. Natl. Acad. Sci. USA 81, 3049 (1984). Additional reports have characterized the intron and demonstrated that the intron is excised by a mechanism analogous to a eukaryotic group I splicing pathway (Ehrenman et al., Proc. Natl. Acad. Sci. USA 83, 5875 [1986]). The excised intron apparently circularizes and is stable, as observed by agarose gel electrophoresis (Belfort et al., Cell 41, 375 [1985]). The structure of the intron of the td gene of bacteriophage T4 is disclosed by Belfort et al., Cold Spring Harbor Symp. Quant. Biol. 52, 181 (1987) and Shub et al., Proc. Natl. Acad. Sci. USA 85, 1151 (1988).
Eukaryotic group I introns are classified into a separate group based on the distinctive secondary structure that the intron RNA can adopt (Michel and Dujon, EMBO J. 2, 33 [1983]). Many eukaryotic group I introns can splice in the absence of proteins and can therefore be excised in heterologous bacterial cells (Cech, Cell 44, 207 [1986]). The td intron of phage T4 is a group I intron, capable of self-splicing and able to be folded into a typical group I structure. Two other phage T4 introns have similar properties (Shub et al., Proc. Natl. Acad. Sci. USA 85, 1151 [1988]). It is this RNA secondary structure that is likely to impart stability to these group I intron RNAs in Escherichia coli (E. coli).
Stable excised introns have been reported in at least two eukaryotic systems. In yeast there are five mitochondrial introns reported to be stable after excision. In Saccharomyces cerevisiae, introns I1, I2, and I5 of the cytochrome oxidase subunit I gene and intron Il of the cytochrome b gene exist in a circular form after excision and are stable (Hensgens et al., J. Mol. Biol. 164, 35 [1983]). In Neurospoa crassa, intron I2 of the ATPase subunit 6 gene exists in a circular form after excision and is stable (Morelli and Macino, J. Mol. Biol. 178, 491 [1984]).
A major aspect of recombinant DNA technology is the production in maximal practical quantities (over-production) of medically, agriculturally or commercially useful protein products in host cells. Briefly, a foreign gene that codes for an important protein is isolated, often by the use or restriction enzymes. The term "foreign" gene designates exogenous DNA that codes for polypeptides not ordinarily produced by the host cell into which the exogenous DNA is placed.
First, the foreign gene is inserted into a cloning vehicle to form a recombinant hybrid. Preferably, a bacterial cloning vehicle is used. A bacterial cloning vehicle is a plasmid or bacteriophage replicon adapted for insertion of foreign DNA. Other cloning vehicles such as those that replicate autonomously in eukaryotic microorganisms, particularly yeast, can be used.
Next, the recombinant hybrid is introduced into the host cell, preferably a bacterial cell such as an E. coli cell. The foreign gene uses the cell machinery to express the protein for which it codes by producing mRNA which subsequently acts as the template for protein production.
There are a number of mechanisms that cells use to regulate the rate of protein synthesis. One such mechanism is the rapid breakdown of mRNAs. Bacterial mRNAs typically have a half-life value of only two minutes.
Despite the fact that a number of techniques are known to the current state of the art for enhancing production of useful gene products in host cells, there is considerable need for further improvements. Consequently, much effort has been expended in developing means for stabilizing the mRNA that produces the protein of interest in order to increase product yields.
For example, Donovan and Kushner, Proc. Natl. Acad. Sci. USA 83, 120 (1986), describe the isolation of E. coli mutants that are defective in ribonucleases that degrade mRNA. Gorski et al., Cell 43, 461 (1985) describe the alteration of the "beginning" (5' leader end) of the mRNA to stabilize the mRNA. Wong and Chang, Proc. Natl. Acad. Sci. USA 83, 3233 (1986) describe the alteration of the "end" (3' end) of the mRNA to stabilize the mRNA. These methods each stabilize the mRNA by a factor of about two to three.
The development of an expression vector based on RNA stabilization has been described (Duvoisin et al., Gene 45, 193 [1986]). However, the stabilization effect is observed only in phage T4-infected cells, making the system very cumbersome. It is well known that phage T4-infected cells lyse and are therefore unable to provide sustained production of useful products.
It can thus be readily appreciated that provision of an RNA stabilization vector that is fully functional in uninfected cells and provides a level of product amplification higher than that provided by the prior art would be a highly desirable advance over the current state of the art in recombinant technology.